The present invention relates to processes for diagnosing and treating Hodgkin""s lymphomas (lymphogranulomatosis) based on the expression of the variant exon v10 of the gene CD44 as the molecular target, agents for these processes and the use of these agents.
The highly glycosylated cell surface protein CD44 is involved in the interaction between cells and the extracellular matrix such as migration and activation of leukocytes in inflammation and immune monitoring, precursor formation of leukocytic and myeloid cells in bone marrow and also in the development of lymphoid organs and the interaction of cells with the extracellular matrix (Lesley et al., 1993, Gxc3xcnthert 1993, Pals et al., 1993, Mackay et al., 1994). The human CD44 gene is made up of at least 19 exons, of which at least 12 which code for the extracellular region are alternatively spliced (Screaton et al., 1992). The CD44 gene is transcribed in a number of normal tissues and carcinomas (Fox et al., 1994). Whereas the standard CD44 molecule (CD44s) is ubiquitously found expressed in epithelial and mesenchymal tissues, the various isoforms produced by alternative RNA splicing are found in very limited distribution (Heider et al., 1993). Some of the variant isoforms are involved in the activation of lymphocytes and occur in conjunction with metastasisation (Mackay et al., 1994, Gxc3xcnthert et al., 1991, Rudy et al., 1993, Koopman et al., 1993). Although the expression of variant CD44 has been shown to have a direct biological role in metastasis formation in carcinoma of the pancreas in rats (Gxc3xcnthert et al., 1991, Seiter et al., 1993), its role in human tumours is as yet unknown.
Various reports have been published showing that certain alternatively spliced forms of CD44 were expressed in human metastatic tumours (Heider et al., 1993 and 1996, Fox et al., 1994, Friedrichs et al., 1995, Kaufmann et al., 1995, Salles et al., 1993, Stauder et al., 1995, Koopman et al. , 1993, Tanabe et al., 1993) Studies of the expression of CD44 in non-Hodgkin""s lymphomas (NHL) concentrated on analysing the so-called lymphocyte homing receptor CD44H or CD44s (Horst et al., 1990a, Horst et al., 1990b, Jalkanen et al., 1991, Mxc3x6ller et al., 1992). Whereas some authors (Horst et al., 1990a, Jalkanen et al., 1991, Picker et al., 1988, Pals et al., 1989, Fujiwara et al., 1993) found a correlation between increased CD44s expression and unfavourable prognosis, other authors (Terpe et al., 1994) could not confirm these findings. Recently, upregulation of CD44v3 and CD44v6 isoforms was found in NHL with unfavourable pathological status (Koopman et al., 1993, Terpe et al , 1994, Salles et al., 1993, Stauder et al., 1995), whilst variant specific CD44-mAbs were used (Mackay et al., 1994, Koopman et al., 1993, Fox et al., 1993).
Various approaches have been developed for making use of the differential expression of variant exons of the CD44 gene in tumours and normal tissues f or diagnostic and therapeutic purposes (WO 94/02633, WO 94/12631, WO 95/00658, WO 95/00851, EP 0531300).
The aim of the present invention was to develop new methods of diagnosing and treating Hodgkin""s lymphomas (lymphogranulomatosis) and preparing agents for such processes.
This aim is achieved by means of the present invention. It relates to processes for diagnosing and treating Hodgkin""s lymphomas (lymphogranulomatosis) which are based on the expression of the variant exon v10 of the CD44 gene as a molecular marker or target. Antibody molecules of corresponding specificity are particularly suitable as vehicles for selectively reaching Hodgkin""s lymphomas in vivo.
Preferred processes are characterised in that an antibody molecule is used which binds specifically to the amino acid sequence SEQ ID NO. 2 (see Sequence Listing).
Other aspects of the present invention are the use of antibody molecules of this kind in the processes according to the invention and agents for performing these processes.
The invention further relates to the use of an antibody molecule which is specific to an epitope within the amino acid sequence which is coded by the variable exon v10 of the CD44 gene, for preparing a pharmaceutical composition for the diagnosis and/or treatment of tumoral diseases. The tumoral disease in question is preferably Hodgkin""s lymphoma (lymphogranulomatosis).
The invention further relates to an antibody molecule which is specific to an epitope within the amino acid sequence which is coded by the variable exon v10 of the CD44 gene for pharmaceutical use. Preferably, an antibody molecule of this kind is characterised in that it binds to SEQ ID NO. 2. It may be, in particular, a monoclonal antibody, an Fab- or F(abxe2x80x2)2-fragment of an immunoglobulin, a recombinantly produced antibody, a recombinantly produced chimeric or humanised antibody or single chain antibody (scFv). Preferably, an antibody molecule of this kind is linked to a radioactive isotope, a radioactive compound, an enzyme, a toxin, a cytostatic, a prodrug, a cytokine or some other immunomodulatory polypeptide.
The nucleic and amino acid sequence of the variant exon v10 of the CD44 gene is known (Screaton et al., 1992, Txc3x6lg et al., 1993). These sequences are shown in the Sequence Listing (SEQ ID NO. 1 and 2). The existence of degenerate or allelic variants is unimportant to the performance of the invention; such variants are therefore expressly included.
The invention may be carried out with polyclonal or monoclonal antibodies specific to an epitope which is coded by the exon v10. The preparation of antibodies to known amino acid sequences can be carried out using methods known per se (Catty, 1989). For example, a peptide of this sequence may be prepared synthetically and used as an antigen in-an immunisation procedure. Another method is to prepare a fusion protein which contains the desired amino acid sequence, by integrating a nucleic acid (which may be prepared synthetically or, for example, by polymerase chain reaction (PCR) from a suitable probe) which codes for this sequence, into an expression vector and expressing the fusion protein in a host organism. The fusion protein, optionally purified, can then be used as an antigen in an immunisation procedure and insert-specific antibodies or, in the case of monoclonal antibodies, hybridomas which express insert-specific antibodies, are selected by suitable methods. Such methods are known in the art. Heider et al. (1993, 1996) and Koopman et al. (1993) describe the preparation of antibodies against variant epitopes of CD44.
However, for the process according to the invention, it is also possible to use antibody molecules derived from poly- or monoclonal antibodies, e.g. Fab- or F(abxe2x80x2)2-fragments of immunoglobulins, recombinantly produced single chain antibodies (scFv), chimeric or humanised antibodies and other molecules which bind specifically to epitopes coded by exon v10. From a complete immunoglobulin it is possible for example to produce Fab-or F(abxe2x80x2)2-fragments or other fragments (Kreitman et al., 1993). The skilled person is also capable of producing recombinant v10-specific antibody molecules. Corresponding methods are known in the art. Recombinant antibody molecules of this kind may, for example, be humanised antibodies (Shin et al., 1989; Gussow and Seemann, 1991), bispecific antibodies (Weiner et al., 1993; Goodwin, 1989), single chain antibodies (scFv, Johnson and Bird, 1991), complete or fragmentary immunoglobulins (Coloma et al., 1992; Nesbit et al., 1992; Barbas et al., 1992), or antibodies produced by chain shuffling (Winter et al., 1994). Humanised antibodies may be produced for example by CDR grafting (EP 0239400). Framework regions may also be modified (EP 0519596). In order to humanise antibodies, nowadays it is possible to use methods such as PCR (cf. for example EP 0368684; EP 0438310; WO 9207075) or computer modelling (cf. for example WO 9222653). It is also possible to prepare and use fusion proteins such as single chain antibody/toxin fusion proteins (Chaudhary et al., 1990; Friedman et al., 1993). The headings xe2x80x9cantibodyxe2x80x9d and xe2x80x9cantibody moleculesxe2x80x9d should include, in addition to polyclonal and monoclonal antibodies, all the compounds discussed in this section as well as other compounds which are structurally derived from immunoglobulins and can be prepared by methods known per se.
For diagnostic purposes, antibody molecules may be linked, for example, to radioactive isotopes such as 131I, 111In, 99mTc or radioactive compounds (Larson et al., 1991; Thomas et al., 1989; Srivastava, 1988), enzymes such as peroxidase or alkaline phosphatase (Catty and Raykundalia, 1989), with fluorescent dyes (Johnson, 1989) or biotin molecules (Guesdon et al., 1979). For therapeutic applications, v10-specific antibody molecules may be linked to radioisotopes such as 90Y, 111In, 131I or 168Re (Quadri et al., 1993; Lenhard et al., 1985, Vriesendorp et al., 1991; Wilbur et al., 1989), toxins (Vitetta et al., 1991; Vitetta and Thorpe, 1991; Kreitman et al., 1993; Theuer et al., 1993), cytostatics (Schrappe et al., 1992), prodrugs (Wang et al., 1992; Senter et al., 1989) or radioactive compounds. The antibody may also be linked to a cytokine or another immunomodulatory polypeptide, e.g. tumour necrosis factor or interleukin-2.
Advantageously, the diagnostic process according to the invention can be used to examine samples from patients, e.g. from biopsies, where there is a suspicion of Hodgkin""s lymphoma (lymphogranulomatosis) or where this has already been diagnosed but the tumour requires more accurate characterisation. Variant CD44 molecules which contain an amino acid sequence coded by the variable exon v10 can be detected at the protein level by means of antibodies or at the nucleic acid level by means of specific nucleic acid probes or primers for polymerase chain reaction (PCR). The invention consequently also relates to antibody molecules and nucleic acids which are suitable as probes or primers for such processes, and the use of these antibodies and nucleic acids for the diagnosis and analysis of Hodgkin""s lymphomas. For example, tissue sections can be investigated immunohistochemically with antibodies using methods known per se. Extracts or body fluids obtained from tissue samples can also be investigated by other immunological methods using antibodies, e.g. by Western blots, enzyme-linked immunosorbant assays (ELISA, Catty and Raykundalia, 1989), radioimmunoassays (RIA, Catty and Murphy, 1989) or related immunoassays. The samples may be investigated qualitatively, semiquantitatively or quantitatively. The expression of the CD44-splice variant v10 in Hodgkin""s disease is associated with aggressive behaviour of the tumour and a high risk of recurrence. This correlates with an advanced stage and poor prognosis of NSHD (nodular sclerosis Hodgkin""s disease).
As well as in vitro diagnosis, antibody molecules with specificity according to the invention are also suitable for in vivo diagnosis of Hodgkin""s lymphomas. If the antibody molecule carries a detectable label, the label can be detected for diagnostic purposes, e.g. imaging the tumour in vivo or for radioguided surgery, for example. For using antibodies conjugated with radioactive isotopes for immunoscintigraphy (imaging), for example, there are a number of procedures on the basis of which the skilled person can perform the invention (Siccardi et al., 1989; Keenan et al., 1987; Perkins and Pimm, 1992; Colcher et al., 1987; Thompson et al., 1984).
Data obtained by detecting and/or quantifying the expression of the variant CD44 epitope v10 can thus be used for diagnosis and prognosis. It may be advantageous to combine such data with other prognostic parameters, e.g. with the grade of tumour.
Antibody molecules with the specificity according to the invention and optionally linked with a cytotoxic agent may advantageously be used to treat Hodgkin""s lymphomas (lymphogranulomatosis). They may be administered systemically or topically, e.g. by intravenous route (as a bolus or continuous infusion), or by intraperitoneal, intramuscular or subcutaneous injection/infusion. Methods of administering conjugated or non-conjugated antibodies, e.g. complete immunoglobulins, fragments, recombinant humanised molecules etc., are known in the art (Mulshine et al., 1991; Larson et al., 1991; Vitetta and Thorpe, 1991; Vitetta et al., 1991; Breitz et al., 1992, 1995; Press et al., 1989; Weiner et al., 1989; Chatal et al., 1989; Sears et al., 1982).
The antibody molecules may be formulated in a manner known per se. For example, they may be present in aqueous solution, optionally buffered with a physiologically acceptable buffer. A solution of this kind may be characterised by the addition of suitable stabilisers and adjuvants. However, the antibody molecules may also be present in the form of a freeze-dried preparation (lyophilisate) which is reconstituted with a suitable solvent, e.g. water, before use.
In a preferred embodiment of therapeutic application, a humanised v10-specific immunoglobulin or an F(abxe2x80x2) 2-fragment thereof is linked with 90Y (Quadri et al., 1993; Vriesendorp et al., 1995), 131I (Juweid et al., 1995; Press et al., 1995; Thomas et al., in: Catty 1985, p. 230-239), 186Re (Breitz et al., 1992, 1995) or another suitable radioisotope and used for radioimmunotherapy of Hodgkin""s lymphomas. For example, an antibody molecule of this kind may be linked with 90Y using a chelating linker such as ITCB-DTPA (isothiocyanatobenzyl-diethylenetriamine pentacetate), whilst a specific activity of 5-20 mCi/mg, preferably 10 mCi/mg should be achieved. This agent can then be administered to a patient with an antigen-positive tumour in a dosage of 0.1 to 1 mCi/kg of body weight, preferably 0.3 to 0.5 mCi/kg of body weight, most preferably 0.4 mCi/kg. When the total quantity of protein to be administered is from 2 to 5 mg this may be given in the form of a rapid intravenous bolus injection. In the case of monoclonal antibodies it may be necessary to mix the agent with an excess (e.g. a ten-fold molar excess) of the non-radioactive antibody before administering it; in this case, the preparation is better administered in the form of an intravenous infusion over a period of 15 minutes, for example. The application can be repeated. The treatment can be backed up by bone marrow transplantation.